Induction of tyrosine hydroxylase expression in rat forebrain neurons

Brain Research, 497 (1989) 117-131 Elsevier

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BRES 14786

Induction of tyrosine hydroxylase expression in rat forebrain neurons Kathleen M. Guthrie and Michael Leon Department of Psychobiology, University of California, lrvine CA 92717 (U.S.A.) (Accepted 14 February 1989) Key words: Olfactory nerve; Olfactory bulbectomy; Tyrosine hydroxylase; Neurotransmitter expression; Neurotransmitter plasticity; Subependymal cell; transmitter phenotype; Forebrain neuron

Olfactory nerve input is required for the normal expression of tyrosine hydroxylase (TH) by dopaminergic neurons in the glomerular region of the rodent main olfactory bulb. To determine whether the olfactory nerve exerts a similar influence on neurons in other brain regions, we performed unilateral bulbectomies in rat pups on postnatal day 5-7 and examined the brains 2-6 months later, after the regenerated olfactory nerve had penetrated the forebrain. Tissue was stained for TH, dopamine fl-hydroxylase (DBH) and olfactory marker protein immunoreactivity. We observed novel TH-immunoreactivity in neurons located in those areas of the adult forebrain which received olfactory nerve fibers, particularly the rostral extension of the subependymal layer. Many of these neurons resembled the periglomerular cells of the olfactory bulb. No cell staining for DBH was observed in these areas, suggesting the possible dopaminergic phenotype of these neurons. Our data indicate that afferent regulation of neurotransmitter expression by the olfactory nerve is not limited to the cells of the olfactory bulb.

INTRODUCTION Mammalian olfactory systems are capable of remarkable anatomical and neurochemical plasticity. The olfactory receptor neurons located in the nasal epithelium are continually replaced throughout an animal's lifetime by proliferative basal cells. These cells differentiate into mature sensory neurons which send their axons to the main olfactory bulb and synapse with the dendrites of mitral, tufted and periglomerular cells in spherical regions of neuropil called glomeruli 2°.47. This ongoing neurogenesis permits reinnervation of the bulb following chemical or surgical lesioning of the olfactory nerve. Such lesions are accompanied by neurochemical changes in the denervated olfactory bulb. For example, levels of olfactory marker protein (OMP), a specific protein expressed by mature olfactory receptor neurons, decrease significantly in the bulb 24'42'43'46. Additionally, enzyme activity, immunoreactivity and m R N A for tyrosine hydroxylase (TH), the rate-limiting

enzyme in the catecholamine biosynthetic pathway, are dramatically reduced in dopaminergic periglomerular and tufted cells 2'13"33'48. Following subsequent reinnervation, these parameters return to normal. The reappearance of normal dopaminergic expression, coupled with the demonstration that immunoreactivity for L-aromatic amino acid decarboxylase (AADC), the second enzyme in the biosynthetic pathway, persists in juxtaglomerular cells during denervation, indicates that these dopaminergic cells survive the manipulation, but reduce their expression of certain catecholaminergic traits when deprived of olfactory nerve input 3. That olfactory nerve fibers regulate the expression of T H in target cells is also suggested by the developmental pattern of TH-immunoreactivity in the bulb. In the mouse, olfactory nerve fibers first reach the bulb on embryonic (E) day 12 (ref. 27). From E l 8 to postnatal (P) day 5, OMP-immunoreactive fibers can be seen extending beyond their normal adult target, the glomerular layer, into the

Correspondence: K.M. Guthrie, Department of Psychobiology, University of California, Irvine, CA 92717, U.S.A. 0006-8993/89/$03.50 © 1989 Elsevier Science Publishers B.V. (Biomedical Division)

118 mitral cell layer. During this time, TH-immunoreactive neurons and processes, located predominantly in the glomerular region in the adult, can also be observed in the mitral cell layer 5. In the rat, the number of TH-immunoreactive glomerular neurons gradually increases after birth at a time when olfactory nerve fiber input to the bulb is also increasing 14'45. Removal of this input in the neonate prevents the developmental appearance of THimmunoreactive bulb cells 45. These studies suggest that in addition to maintaining normal TH expression in bulb neurons, the olfactory nerve is responsible for initiating this expression during development. Olfactory bulbectomy concomitantly severs the olfactory nerve and removes its normal target. Approximately one month after neonatal bulbectomy, the regenerating nerve penetrates spared portions of the ipsilateral forebrain and olfactory axons can be seen coursing within portions of the anterior olfactory nucleus (AON), prepyriform and frontal cortices, and along the subependymal layer of the lateral ventricle ~8'19'55'56. Within these areas, the sensory terminals ramify in areas of neuropil which, at two months, resemble the glomeruli of the olfactory bulb. Graziadei's group has demonstrated that in many of these glomeruli, synaptic contacts are made between these terminals and the dendrites of large, argyrophilic neurons which resemble mitrai and tufted cells 19"22. These large cells are often observed in close proximity to the glomerular structures, while smaller surrounding neurons, similar to bulb periglomerular cells, are usually lacking, except when glomerularization occurs in portions of the subependymal layer 1s'19'22'56. The olfactory bulb is itself a forebrain structure and as the olfactory nerve is clearly capable of both regulating bulb catecholaminergic expression and organizing forebrain tissue, the present study was designed to investigate whether the olfactory nerve is capable of influencing the transmitter chemistry of forebrain neurons. MATERIALS AND METHODS

Animals Male Wistar rats from our breeding colony (Hilltop Lab Animals, Scottsdale, PA) were housed in polypropylene cages (48 x 25 × 16 cm) in a 12-h

light/dark cycle with rat chow and water available ad libitum. All animals appeared healthy throughout the study. On PI (day of birth = P0), litters were culled to 9 male pups. Unilateral bulbectomy was performed under cold anesthesia on P5-P7. With a sharp scalpel, the skin overlying the skull was incised along the midline to expose the frontal bones. In pups, the position of the olfactory bulbs can be seen through the skull. A sharp no. 11 scalpel was used to carefully cut and lift a flap of frontal bone to expose the left bulb. A fine microsurgical knife was passed between the frontal lobe and the left bulb to sever the bulb's connections with the forebrain. With the aid of a dissecting microscope, the left bulb was aspirated using a blunted 22-gauge needle connected to a vacuum line. The needle was angled ventrally and caudally to ensure removal of the most posterior portions of the bulb, and in many cases small areas of the forebrain were also removed. The bone flap was repositioned over the resulting cavity and the skin wound closed with a cyanoacrylic glue. Shamoperated littermates underwent frontal bone cuts, leaving both bulbs intact. For all animals, bleeding was minimal under the cold anesthesia. The pups were placed on a warming pad to recover prior to returning them to the dams. The entire surgicalrecovery period took about 15 min and survival rate was excellent (90%). Animals were sacrificed 2-6 months later and the tissue examined for TH, dopamine fl-hydroxylase (DBH) and OMP immunoreactivity. Results are based on comparisons of 6 control and 10 experimental animals.

Immunocytochemistry Animals were deeply anesthetized with Nembutal and transcardially perfused with 100 ml of 0.05 M phosphate-buffered saline (PBS) followed by 500700 ml of 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4). The brains were postfixed for 12 h and stored in 0.1 M phosphate buffer containing 20% sucrose overnight (4 °C). Sagittal sections were cut at 25-30/~m on a cryostat and collected into 0.1 M phosphate buffer. Free floating sections were blocked for 30 min in a 3% solution of bovine serum albumin (BSA) in 0.05 M Tris buffer, rinsed, and incubated overnight at 4 °C in a 1:1000 dilution of primary antibody in 0.05 M Tris and 0.15 M NaCI

119 containing 0.7 mg/ml BSA and 0.3% Triton (pH 7.4). For immuno staining, sections were incubated in polyclonal antibody to TH (rabbit; Eugene Tech Inc., Allendale, NJ; Pel-Freez Clinical Systems, Brown Deer, WI), D B H (rabbit; Eugene Tech Inc., Allendale, N.J.) or OMP (goat; kindly provided by Dr. EL. Margolis). Control sections were incubated with non-immune rabbit serum (1:1000). For immunoperoxidase staining, sections were then rinsed in PBS and incubated at room temperature for 1 h in the appropriate biotinylated secondary antibody provided in the Vectastain kit (Vector Laboratories, Burlingame, CA). The tissue was rinsed again, followed by incubation for 1 h at room temperature in avidin-HRP biotin complex according to the kit's instructions. After rinsing, the reaction product was developed in 3,3"-diaminobenzidine in 0.1 M phosphate buffer (33 mg/100 ml) containing 0.03% H 2 0 2. Sections were mounted on gelatin-coated slides, dehydrated and coverslipped. Following microscopic examination, sections of interest were counterstained with Cresyl violet. Photographs were taken on a Nikon HFX microscope. For fluorescent double-labeling, the tissue was first incubated in OMP antibody overnight, rinsed, then incubated in T H antibody for 12 h. After washing, sections were incubated at room temperature in a 1:50 dilution of fluorescein isothiocyanate (FITC)-conjugated swine anti-goat IgG (ICN ImmunoBiologicals, Lisle, IL) for 30 min, rinsed, then incubated in a 1:20 dilution of rhodamine-conjugated donkey anti-rabbit IgG (Chemicon, E1 Segundo, CA) for 1 h. Following rinsing, sections were mounted on gelatin-coated slides and allowed to air dry before being coverslipped with phosphate-buffered glycerol (1:9). Photographs were taken on an Olympus BH 2 epifluorescence microscope equipped with EY455 and EO515 barrier filters for exclusive viewing of FITC and rhodamine fluorescence. RESULTS In experimental animals, typical foward displacement of the frontal cortex into the cavity vacated by the olfactory bulb occurred. In one animal the remaining right bulb had shifted toward the midline. Although at the time surgery was performed, bulb-

ectomy appeared complete, about a third of our experimental animals retained bulb fragments visible at the light microscopic level. Those animals which retained greater than about 15% of their bulb tissue

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Fig. 1. A: low power photomicrograph of a sagittal section through the forebrain of a rat sacrificed 5 months after complete unilateral bulbectomy. The tissue is stained with antiserum to OMP. Olfactory nerve fibers have tracked along the rostral projection of the subependymai layer, stopping just short of the caudate. Fibers can also be seen ventrally in the olfactory peduncle. B: camera lucida drawing of an adjacent section showing the distribution of THqmmunoreactive neurons (filled dots). The cells in area 1, in the caudal portion of the subependymal layer, are shown in Fig. 3A. From here, the band of TH-stained ceils extends anteriorly to the rostral edge of the remaining forebrain. Within this most rostral region (area 2), large numbers of granule cells, continuous caudally with the subependymal layer, can be seen in sections stained with Cresyl violet. The immunoreactive cells in area 3 are located in the superficial layers of the olfactory peduncle. CPu, caudate putamen; FC, frontal cortex; Iv, lateral ventricle; Tu, olfactory tubercle. Bars = 1 ram.

120 did not exhibit olfactory nerve penetration into the forebrain. Those animals subjected to more complete bulbectomies displayed clear evidence of ipsilateral forebrain penetration by the olfactory nerve in several areas. Bundles of OMP-immunoreactive fibers, many of which aggregated into glomerularlike stuctures, were observed in frontal cortex and in ventral portions of the olfactory peduncle and AON. Nerve penetration was especially pronounced along the rostral projection of the subependymal layer of the lateral ventricle, and in some cases stopped just short of the caudate nucleus (Fig. 1A). In sections stained with Cresyl violet, the rostral border of the remaining forebrain could be seen to contain an area of small, densely packed granule cells, continuous caudally with the subependymal layer. Glomeruli tended to be concentrated within this anterior cell region and were less frequently encountered caudally where the olfactory nerve fibers formed a more uniform tract along the subependymal cell migratory route. Once this cell layer was contacted, the nerve fibers appeared to track along it, diverging only rarely. Nerve penetration into the subependymal layer was usually not apparent when significant portions of the AON and peduncle were removed during the bulbectomy. When these ventral forebrain areas were lacking, glomerularization occurred in more dorsal regions of the forebrain, i.e. frontal cortex. None of our experimental animals displayed evidence of anomalous innervation of the contralateral bulb. In adjacent sections, TH-immunoreactive neurons could be seen in those forebrain areas which contained OMP-positive fibers (Fig. 1B). Both of the TH antibodies gave similar results. Only experimental animals which clearly displayed olfactory nerve penetration into the forebrain as judged by OMP

staining exhibited TH-immunoreactive forebrain cells and then only in areas corresponding to this ingrowth. No such cell staining was observed in the contralateral forebrain (Fig. 3B), in sections incubated in non-immune serum, in sham-operated control tissue or in experimental brains devoid of OMP-immunoreactive fibers. Most striking was a stream of TH-positive cells distributed along the rostral projection of the subependymal layer, from the rostral edge of the spared forebrain to the level of the caudate putamen (Figs. 1B, 2B and 3A). These neurons were small, ranging from 7 to 12 ~m in diameter and most often appeared bipolar, with their processes extended parallel to the subependymal cell stream (Fig. 3C). Other neurons were seen as far as 400/~m from this main group, but these cells were rare. In most instances TH staining revealed a single process, but some cells exhibited one or two additional processes which emerged from the soma or from the base of the main trunk (Fig. 3D). Often, the stained neurons within the subependymal zone projected their processes in parallel but opposite directions. Somas were usually ovoid, and appeared elongated along the axis of the cell stream. Caudally, the subependymal cell band widened as it approached the head of the caudate, and TH-positive cell bodies were distributed further from the main axis of the group with their processes often directed toward it (Fig. 3A). Those neurons located at some distance from the main axis had somewhat rounder cell bodies. Occasionally, patches of fine TH-positive terminals could be identified within the wider zone near the caudate and stained processes were evident along the entire length of the rostral subependymal projection. The great majority of subependymal cells did not exhibit immunoreactivity for TH and neurons which stained were surrounded by many others which did not.

Fig. 2. Pairs of fluorescence photomicrographs (A and B; C a n d D) of sagittal sections illustrating the presence of TH-immunoreactive neurons in areas of the forebrain innervated by OMP-immunoreactive fibers. A and C were photographed with a blue filter for visualization of FITC fluorescence; B, D, E and F were photographed with a green filter for visualization of rhodamine fluorescence. A: OMP-immunoreactive fibers coursing along the rostral extension of the subependymal layer. B: same section showing TH-immunoreactive neurons distributed along the subependymal layer. The cells in the lower right are projecting their processes towards an OMP-labelled glomerulus located ventrally (bottom, not shown) in the olfactory peduncle. C: OMP-immunoreactive glomerulus in the AON. D: same section showing a TH-immunoreactive cell in the AON sending a process toward this glomerulus. E: another TH-immufforeactive cell (thin arrow) located further caudally. The head of the caudate (broad arrow) is included as a reference point. This neuron displayed two prominent processes, each directed toward one of two glomeruli located ventrally (bottom, not shown), F: high power photomicrograph of same cell. A second stained cell can be seen to the right, out of the plane of focus. Bars for A - E = 100 #m. Bar for F = 50 prn.

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Fig. 3. TH-immunoreactive cells in the forebrain of a unilaterally bulbectomized animal. A: low power photomicrograph of the area rostral to the caudate in the ipsilateral forebrain. B: same area in the contralateral forebrain. TH fiber staining can be seen in the caudate, but no cell staining occurs. C,D: high power photomicrographs of stained cells in the subependymal layer. Most cells extend at least one process which runs parallel to the cell stream; others exhibit more than one process (arrow in C. and D). CPu. caudate putamen. Bars in A - C = 50 l~m. Bar ill D = 20 , m .

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Fig. 4. Photomicrographs of TH-imrraunoreactive neurons clustered around glomeruli in the rostral granule cell group. A: sagittal section stained for TH-immunoreactivity showing cells surrounding a glomerulus (GI). B: similar section counterstained with Cresyl violet to show surrounding granule cells (out of the plane of focus). Darkly stained TH-immunoreactive cells border the glomerulus and can be seen sending processes into it. Bars = 50/~m.

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Fig. 5. A: small TH-stained cells, some with processes (arrow), in the superficial layers of rostral frontal cortex. B: TH-stained section counterstained with Cresyl violet. A small immunoreactive neuron (arrow) can be seen in the A O N , surrounded by larger cells which do not stain with TH antibody. C: similar section at lower magnification showing a glomerulus (GI) within the AON. Ventral is left. The subependymal layer (not shown) is to the right. The thin arrows indicate TH-immunoreactive cells within the AON. Another immunoreactive cell is located at the border of the glomerulus, with a process directed into it (broad arrow). Bars 50/zm. =

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Fig. 6. Cell staining for TH- and DBH-immunoreactivities in adjacent sections from a bulbectomized animal. The asterisks mark the location of a blood vessel visible in both (A) and (B). A: TH-immunoreactive neurons within the subependymal layer. B: adjacent section processed for DBH-immunoreactivity. No cell staining is apparent. C: DBH-immunoreactive cells in the ipsilateral locus coeruleus of the same animal. Bars = 100/~m.

Anteriorly, clusters of small TH-immunoreactive neurons, continuous with the rostral extension of the subependymal group, were observed within the dense aggregation of granule cells. They ranged in size from 9 to 14 p m in diameter and once out of the main axis of the subependymal layer the cell bodies appeared rounder and branching processes were frequently observed. Cells often had more than one process emerging from the soma, but these could not be identified as either axons or dendrites with certainty. Processes were no longer extended in parallel directions and many were directed toward glomeruli located within the rostral granule cell group. Some of the stained neurons were located along the borders of these glomeruli, although in fewer numbers than normally seen in the olfactory bulb, and stained terminals were apparent within the glomerular structures (Fig. 4A,B). In two experimental animals, small TH-positive cells were also seen in frontal cortex (Fig. 5A). The cortical areas containing these cells exhibited staining for OMP in adjacent sections. Large portions of the ventral peduncle and A O N were lacking in these animals and nerve penetration along the remaining sub-

ependymal layer was minimal. Overall, the forebrains of these animals contained fewer TH-immunoreactive neurons than did those animals in which sensory fiber ingrowth occurred primarily in the ventral forebrain and subependymal zone. No cell staining for T H was seen in two other animals which exhibited olfactory nerve penetration into frontal cortex. Ventrally, in the superficial layers of the olfactory peduncle and the AON, TH-positive neurons were observed in areas heavily stained for OMP in adjacent sections (Fig. 2C,D). These cells ranged from 9 to 15 /~m in diameter and were similar to those seen in the subependymal layer, but more frequently displayed multiple and branching processes (Fig. 2E,F). These processes were often directed toward glomerular stuctures which could be identified in TH-stained sections by the presence of a fine network of labeled terminals. These glomeruli were not as heavily labeled for TH as were their normal counterparts in the olfactory bulb, and in most instances were also surrounded by fewer TH-positive cells. Stained neurons were occasionally seen at intermediate locations between the sub-

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Fig. 7. Photomicrograph of a sagittal section through the remaining bulb tissue of an animal that was partially bulbectomized. Tissue is stained for TH-immunoreactivity and counterstained with Cresyl violet. TH-positive cells (arrows) can be seen near both an ectopic glomerulus (gl) and the subependymal layer (SE) of the remnant olfactory bulb (bordered by dashed lines). Bar = 50 pm.

ependymal layer and the ventral peduncle. Some of these cells appeared within the deep layers of the A O N surrounded by large neurons which did not stain for TH (Fig. 5B,C). In other animals the subependymal and peduncle cell groups appeared completely separate. No cell staining for DBH-immunoreactivity was observed in the forebrains of any of the experimental animals. Areas containing TH-immunoreactive cells appeared devoid of D B H staining in adjacent sections, while cell staining for D B H in the locus coeruleus ipsilateral to the lesion appeared normal (Fig. 6). Based on their size, morphology, and relation to glomerular structures, many of the TH-stained cells we observed in the forebrains of bulbectomized animals resemble periglomerular cells of the olfactory bulb. A few of the larger neurons, particularly

those located at some distance from glomeruli and with processes projecting toward them, resemble tufted cells. None of the TH-positive cells we observed were as large as the neurons previously described in association with forebrain glomerular structures 22 and presumably these large cells do not express TH in response to olfactory nerve ingrowth. Partial bulbectomy results in disruption of the normal laminar organization of the bulb and the formation of glomeruli within all layers of the remaining tissue 21. In those animals which retained substantial bulb fragments, we observed TH-immunoreactive neurons within the granule cell layer, surrounding ectopic glomeruli (Fig. 7). In the transition zone between the granule cell layer and subependymal layer of the bulb, small stained cells, similar to those seen in the subependymal layer of completely buibectomized animals, were sometimes

127 observed in close proximity to these glomeruli, but only within the boundaries of the remaining bulb and never further caudally. DISCUSSION Regulation of normally expressed neuroactive substances in the brain by a variety of environmental factors is well documented 9A7'25'41"59. Although novel TH expression is exhibited by embryonic cortical neurons transplanted into adult cortex or maintained in culture 29"49, this is the first demonstration of induced TH expression in resident cells of the adult forebrain. We feel that the mechanisms underlying this response constitute an induction of this transmitter trait for several reasons. Although a small number of neurons in rat frontal cortex express TH-immunoreactivity transiently at day El8, by E21 this population disappears 58. Transient immunoreactivity for T H has also been detected in fibers within the embryonic ventricular zone but this immunoreactivity also disappears before birth 57. Another transient cell population has been described in the dorsal telencephalon of the rat from P8 to P24, but none of these cells are contained within the subependymal layer or the AON 8. Following neonatal olfactory bulbectomy, it takes approximately 3 weeks for the regenerating olfactory nerve fibers to reach and penetrate the remaining forebrain 18'19"56. By this time, detectable expression of TH in the aforementioned brain areas has disappeared. While olfactory nerve ingrowth may provide some signal that allows re-expression of TH in some rat forebrain neurons which previously expressed the enzyme, this still constitutes an induction rather than a maintenance of the trait. Alternatively, this ingrowth may induce de novo T H expression in responsive neurons. This is likely to be true of subependymal cells in particular as expression of TH-immunoreactivity by this particular cell group has never been demonstrated. As with all studies employing immunohistochemistry, our interpretation is limited by the resolution of the technique. It is possible that the occurrence of TH-immunoreactivity which we have observed in rat forebrain neurons represents an induced increase in TH synthesis in neurons which express the protein at levels normally too low to be detected. While TH-immunoreactive neurons have

not previously been detected in the normal adult rat forebrain, such cells have been demonstrated in the basal forebrain of the monkey 35, a species difference that could reflect either actual differences in forebrain cell phenotype or dramatically different levels of TH expression. Interestingly, the TH-positive cells described in the monkey brain tended to be concentrated near the olfactory tract and in the olfactory tubercle, but no cell staining was seen in the subependymal layer35. The factor or factors involved in the peripheral afferent regulation of transmitter traits in bulb and forebrain neurons is not known. Proximity to olfactory nerve fibers is not in itself sufficient for normal dopaminergic expression by bulb cells. Olfactory deprivation resulting from neonatal occlusion of the naris produces a reduction in both TH-immunoreactivity and dopamine levels, suggesting that afferent activity per se or coincident release of trophic substances maintains normal bulb dopaminergic expression 6'1°. We do not yet know if functional contacts are established by olfactory nerve fibers and TH-positive forebrain neurons in bulbectomized animals. The effects of olfactory nerve input on bulb transmitter synthesis is not limited to the dopaminergic system; deafferentation also reduces substance P- and CCK--immunoreactivities in juxtaglomerular neurons of the hamster and rat, respectively38'54. However, immunoreactivity for ~-aminobutyric acid (GABA) in the glomerular region remains unaltered by deafferentation 4, indicating that different phenotypic populations within the bulb are subject to different regulatory influences. As a subpopulation of glomerular neurons has been shown to contain both GABA- and TH-immunoreactivities 16, differential afferent regulation of colocalized substances within a single cell population is another possibility. Whether or not these effects are due to direct actions by the olfactory nerve fibers on responsive postsynaptic targets or via indirect actions on other bulb elements is not known. The effects of centrifugal inputs on bulb transmitter expression have not been examined. The presence of TH-immunoreactive neurons in the subependymal layer of bulbectomized animals and in the granule cell layer of those only partially bulbectomized, is particularly interesting from a

128 developmental standpoint. The granule cell layer of the rodent olfactory bulb, which does not receive direct input from olfactory nerve fibers, normally contains only a small number of scattered THimmunoreactive cells of the short axon type 1~, although the turtle bulb contains a substantial number of TH-positive neurons in the outer part of the granule layer 23. The majority of granule and periglomerular cells appear to be GABAergic 52. Although these different classes of mature interneutons appear morphologically and neurochemically different, they may represent a developmental continuum, cell phenotype being determined by the environments encountered during migration through and arrival at a particular location in the bulb. In the rat bulb, most of the major interneurons, the periglomerular, granule, and short axon cells, are generated simultaneously from the subependymal zone of the bulb during the first few postnatal weeks 7'26. A weak temporal gradient exists, with development of the periglomerular population slightly leading that of the granule cells, followed by their migration past mitral and tufted cells to their final positions in the glomerular layer 726. Proximity to olfactory nerve fibers clearly provides some signal which encourages development of the dopaminergic chemical phenotype, and may foster the development of periglomerular morphology as well. Several laboratories have demonstrated that many periglomerular cells immunoreactive for TH are also immunoreactive for GABA, although there appears to be considerable variability in the reported proportion of cells which do SO 16'36~37. This would be consistent with the acquisition of dopaminergic/ periglomerular cell traits by cells which may initially express GABAergic/granule cell traits, as the cells are increasingly influenced by developing olfactory nerve fibers. Periglomerular cells which develop the dopaminergic phenotype may retain the ability to express GABA. Interestingly, the number of TH-immunoreactive neurons in the glomerular region continues to increase with age, well past the period during which juxtaglomerular neurons are generated 44'45, and after a reported decline in the periglomerular population 15. While late cell migration into the glomerular region could account for this increase, it may instead reflect a parallel increase in the olfactory

receptor neuron population which continues into adulthood 2s. If glomerular neurons compete for olfactory nerve factors which promote dopaminergic expression, the continual expansion of afferent input to the bulb may increase the availability of such factors, permitting more cells to express TH. This might account for some of the variability reported in colocalization studies from different laboratories. Clearly further studies are needed to investigate possible developmental changes in the proportions of periglomerular, granule and tufted cells which express putative transmitter substances. The apparent phenotypic plasticity of the subependymai cells themselves is perhaps not surprising since this rostrally migrating population maintains its neurogenetic capability well into adulthood by continuing to generate new, 'immature' cells 1. Most of these cells migrate into the granule cell layer of the olfactory bulb, although there is some evidence that anterior portions of the neocortex also acquire small numbers of new cells from this group 1'31'32'34. Young, relatively undifferentiated neurons are typically more malleable than mature neurons 3°'5°, and this population may provide a particularly useful system for the study of central transmitter plasticity in much the same way that neural crest derivatives have been employed to study the peripheral nervous system 12"39"4°'51. The apparent plasticity of subependymal cells, as demonstrated here, may in part be responsible for the forebrain's capacity to accept ingrowing sensory fibers. The fact that not all subependymal cells produce TH when exposed to olfactory nerve fibers may indicate phenotypic heterogeneity within the population or dependence on developmental stage. Only postmitotic central neurons express catecholaminergic traits and subependymal cells which do not exhibit TH-immunoreactivity may still be mitotically active s3. The subependymal population appears to be the source of the TH-positive cells we observed in the rostrai granule cell group; we had the distinct impression that these TH-stained cells had migrated rostrally from more caudal regions of the subependymal layer. Graziadei and co-workers similarly noted what appeared to be a migration of subependymal cells toward newly formed forebrain glomeruli TM. The morphology of the immunoreactive cells in the anterior two-thirds of the subependymai

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layer also suggests active migration. This was particularly true of those neurons which extended processes parallel to the direction of the subependymal cell migratory stream. In contrast, the distribution and morphology of some of the stained neurons located in caudal portions of the subependymal zone near the head of the caudate suggested they were not migrating. They tended to be located outside the main axis of the subependymal cell stream and displayed more complex process arborization. The migration of these cells to more anterior locations may have been arrested by contact with ingrowing olfactory nerve fibers. The origin of the TH-immunoreactive neurons in the olfactory peduncle and frontal cortex is not certain. While they may be cells which have migrated from the subependymal zone, they may instead be forebrain neurons which have been modified in situ by olfactory nerve ingrowth. Examination of tissue from several of the experimental animals left us with the impression that the cells had migrated from the subependymal zone. A few immunoreactive cells were located in the superficial layers of tissue in the area lying between the stained cells of the frontal cortex and the stained cells of the rostral subependymal layer. Immunoreactive neurons were also observed at intermediate locations between the ventral peduncle and the subependymal layer. If the TH-positive neurons we observed in the AON, olfactory peduncle and frontal cortex did reach their final locations by moving out from the subependymal zone, then the presence of olfactory nerve fibers in these regions may have served to REFERENCES 1 Altman, J., Autoradiographic and histological studies of postnatal neurogenesis. IV. Cell proliferation and migration in the anterior forebrain, with special reference to persisting neurogenesis in the olfactory bulb, J. Comp. Neurol., 137 (1969) 433-458. 2 Baker, H., Kawano, T., Margolis, EL. and Joh, T.H., Transneuronal regulation of tyrosine hydroxylase expression in the olfactory bulb of mouse and rat, J. Neurosci., 3 (1983) 69-78. 3 Baker, H., Kawano, T., Albert, V., Joh, T.H., Reis, D.J. and Margolis, EL., Olfactory bulb dopamine neurons survive deafferentation-induced loss of tyrosine hydroxylase, Neuroscience, 11 (1984) 605-615. 4 Baker, H., Towle, A. and Margolis, EL., Differential afferent regulation of dopaminergic and GABAergic neurons in the mouse main olfactory bulb, Brain Research, 450

redirect their normal migratory path, in addition to influencing their neurochemistry. We have demonstrated that olfactory nerve ingrowth is capable of inducing the expression of the catecholamine biosynthetic enzyme, TH, in neurons of the rat forebrain which normally lack detectable expression of this protein. The functional transmitter phenotype of the TH-immunoreactive neurons we observed remains unknown, although the lack of DBH-immunoreactivity suggests they do not synthesize norepinephrine. Further study is needed to determine if the T H protein expressed is catalytically active, and if A A D C is synthesized. Whether or not these cells actually synthesize, store and release dopamine, the demonstrated ability of olfactory receptor neurons to influence the neurochemistry of cells in both the bulb and the forebrain makes this a particularly interesting system for the study of both phenotypic plasticity and the factors which regulate neurotransmitter expression in the central nervous system. ACKNOWLEDGEMENTS We would like to thank Dr. Frank Margolis for the generous gift of OMP antibody and Dr. Christine Gall for helpful comments on the manuscript. This work was supported by Grant NS 21484 from NINCDS to M.L., who holds Research Development Award MH 00371 from NIMH. K.M.G. was supported by predoctoral training Grant MH 09635 from NIMH. Support was also provided by an award from the Pew Charitable Trust. (1988) 69-80. 5 Baker, H., Development of olfactory marker protein (OMP) in the receptor epithelium and brain in the mouse as well as its relationship to tyrosine hydroxylase (TH) expression, Soc. Neurosci. Abstr., 14 (1988) 1168. 6 Baker, H., Neonatal olfactory deprivation results in a dramatic reduction of tyrosine hydroxylase levels in adult rat main olfactory bulb, Chem. Senses, in press. 7 Bayer, S.A., [3H]Thymidine-radiographic studies of neurogenesis in the rat olfactory bulb, Exp. Brain Res., 50 (1983) 329-340. 8 Berger, B., Verney, C., Gaspar, P. and Febvret, A., Transient expression of tyrosine hydroxylase immunoreactivity in some neurons of the rat neocortex during postnatal development, Dev. Brain Res., 23 (1985) 141-144. 9 Black, I.B., Adler, J.E., Dreyfus, C.F., Jonakait, G.M., Katz, D.M., LaGamma, E.E and Markey, K.M., Neurotransmitter plasticity at the molecular level, Science, 225

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